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June 1983]
Neodymium-Yttrium Aluminium Garnet Laser Destruction of Nonsensitized and
Hematoporphyrin Derivative-sensitized Tumors1
Thierry Patrice, Marie-Françoise Le Bodic,2 Louis Le Bodic, Thierry Spreux, Gérard Dabouis, and
Luc Hervouet
DéparlementLaser CHU Nantes [T. P., L L B., T. S., G. D., L H.] and Laboratoires d'Anatomie Pathologique A et B [M-F. L. B.], Facultéde Médecine,1 rue Gaston Veil,
44035 Nantes, France
ABSTRACT
The injection of hematoporphyrin derivative (5 mg/kg ¡.v.)
followed 24 hr later by a neodymium-yttrium aluminium garnet
laser irradiation shows the destruction of CX1 tumors grafted on
nude mice. This acidophilic necrosis occurred with a significantly
increased frequency in tumors treated by hematoporphyrin de
rivative injection and irradiated with the neodymium-yttrium alu
minium garnet laser as compared with noninjected but irradiated
tumors or with injected tumors irradiated with sunlight.
On the basis of our data, it seems difficult to maintain the
hypothesis of singlet oxygen production as the only mechanism
of the phenomenon. Further studies will be necessary to explain
the necrosis that we observed.
INTRODUCTION
Envisaged in the 1900s by Tappeiner et al. (13), the affinity of
hematoporphyrin for neoplastic tissues has been confirmed by
Figge ef al. (5) and Lipson et al. (9) and was demonstrated by
Rasmussen-Taxdall ef al. (12), Gregorie ef al. (6), and Kelly and
Snell (7) using HPD.3 Later, the oxidizing properties of HPD
under different non-ionizing rays were demonstrated regardless
of the wavelength used (mercury vapor lamps or xenon arc
lamps) or whether or not it was filtered (1, 2). These studies led
Dougherty ef al. (3) to use heliunrneon or dye lasers as sources
of excitation of HPD. This led us to study the effects of photocoagulation by the Nd-YAG laser on human colonie adenocarcinoma-type tumors grafted in the hip of the nude mouse after
sensitization of the tumor tissue by HPD.
The aim of the study was to evaluate under these conditions
the efficacy of the Nd-YAG laser and to study the lesions created
in order to determine whether they were specific or similar to
those seen in control series.
MATERIALS
AND METHODS
Materials
Male albino nude mice were used. A human colonie adenocarcinomatype tumor (C x 1) was grafted by bilateral injection of 0.2 ml of crushed
tumor in solution in culture medium.
PD was prepared using the method of Gregorie ef al. (6) and Lipson
ef al. (9). Hematoporphyrin hydrochloride (Roussel) was dissolved in
acetic acid:sulfuric acid (9:1). The mixture was left at room temperature
for 12 hr and was then filtered and neutralized by addition of a 3%
sodium acetate solution until a pH of 6 was obtained. The precipitate
collected by filtration was washed until a neutral washing water was
obtained, and it was then dried and stored at -20° in a solid state and
1This work has been supported by C. N. E. H. (French Ministry of Health).
1 To whom requests for reprints should be addressed.
3 The abbreviations used are: HPD, hematoporphyrin derivative; Nd-YAG, neo
dymium-yttrium aluminium garnet.
Received May 18,1982; accepted February 8,1983.
2876
in darkness. The solution for injection was prepared by mixture of one
part of HPD and 50 parts of 0.1 N soda solution left at room temperature
for 1 hr. The pH was adjusted at 7.2 to 7.4 with the addition of
hydrochloric acid solution and made isotonic by the addition of sodium
chloride. The final 5-mg/ml solution was sterilized by filtration through a
Millipore filter and kept in darkness at -20° until used.
The energy was produced
by a Nd-YAG
laser (Cilas-Marcoussis
Laboratory, 91460, France) emitting in the near IR (1.06 //m). This laser
is a solid-state laser commonly used in our medical applications of laser
therapy. A mechanical device held the tip of the fiber constantly at 5 mm
from the target. A system combining a TRG 101 thermopile and a TRG
102 energy meter (Control Data Corp., Yellow Springs Institute, Colo.)
was used to measure the power at the end of the fiber.
The visualization beam of the helium:neon laser was cut off throughout
the experiment.
Methods
All of the mice received a tumor graft on the first day of the experiment.
Groups were constituted as follows.
Experimental Series (Group I; 14 Tumors). On the ninth day, the
mice received an injection of HPD (5 mg/kg). On the 10th day, following
skin incision (to prevent secondary skin necrosis caused by laser heating)
which did not interfere with the deep vascular supply of the tumor, the
tumors received a series of Nd-YAG laser exposures. The incision was
then stitched up to avoid septic contaminations. On the 12th day, the
mice were sacrificed, and the tumors were removed for histopathological
examination.
Comparative
Control Series (Group II) with the Following
Subgroups. In Group II-A, 4 tumors were removed on the 12th day, the
mice having received one tumor graft only; in Group II-B (8 tumors), on
the ninth day, the mice received an injection of HPD (5 mg/kg). They
were sacrificed on the 12th day for examination; in Group II-C (8 tumors),
the mice received a similar injection of HPD, but on the 10th day, after
skin incision, the tumors were exposed to sunlight through the window
glass for 5 min. This group was formed to show that the observed effect
of Group I was not linked to the surrounding light. These mice were
sacrificed on the 12th day; in Group II-D (8 tumors), the mice did not
receive an injection of HPD, but the tumors were treated with the laser
on the 10th day and removed for examination on the 12th day.
The energy level of the laser has been controlled for each tumor of
Groups I and II-D. For every shot, the power was 45 watts, and time
exposure was 1.5 sec (Table 1).
The same experimental conditions used to obtain sample material,
anesthetic techniques, and darkness were applied to all of the different
groups. All of the mice were sacrificed on the 12th day for histopatho
logical examination of the tumor. The latter was fixed in 12% formol for
24 hr, cut along its long axis, and mounted in its entirety in several
paraplast blocks before being studied by numerous serial sections
stained with hematoxylin:eosin:safran.
RESULTS
Group I (cf. Fig. 1). The 14 tumors which photocoagulated
after sensitization with HPD showed necrosis affecting approxi
mately 90% of the total tumor mass. In 10 of them, there was
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VOL. 43
Laser Destruction of Non- and HPD-sensitized Tumors
total acidophilic necrosis leaving no detectable tissue or cellular
structure. In 3 tumors, there was necrosis although it was not
acidophilic, with loss of glandular arrangement, disappearance
of cell borders, and retraction of the nuclei when they could still
be identified.
At the extreme periphery of the tumor, and in particular at
depth, in 12 of 14 cases, rare neoplastic glandular tubes per
sisted, representing approximately 10% of the total tumor mass.
On the surface and laterally, at the level of the cutaneous tissue,
edema, congestion, and moderate inflammatory infiltrate were
observed.
In depth and beyond the tumor, the muscular layer was
affected in more than one-half of all cases.
Group II-A (cf. Fig. 2). Upon histological examination, these 4
control tumors, varying from 7 to 10 mm in diameter, were found
to be weakly mucosecreting adenocarcinomas. Tumor spread
developed in the s.c. tissue. The arrangement was frankly glan
dular and was sometimes polyadenoid with neoplastic tubules
lined by an epithelium rich in cytological and nuclear abnormalities
Table 1
Tumor size and dose exposures in Groups I and II-D
tumor diame
ter (mm)86.8Av.
Group I
Group II-DAv.
no.shots5.4
of
4.8Av.
(J)364.5
energy
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Fig. 2. Group II-A, control tumor:human colonie adenocarcinoma
(Cx1). Hematoxylin, eosin, and safran; A, x 3.7; B, x 250.
type tumor
and with a high mitotic index. There were few fibroblastic stroma.
There was neither localized abscess formation nor necrosis apart
from one case where there was a very limited area of necrosis.
Group II-B (cf. Fig. 3). In these mice, which had received
HPD, the nonphotocoagulated tumors were the site of rare areas
of scanty punctiform necrosis with persistence of >90% of the
total tumor mass. However, in 2 cases, there was an area of
complete acidophilic necrosis.
Group II-C (cf. Fig. 4). These mice received an injection of
HPD. These tumors were exposed to sunlight 24 hr later.
Five tumors were the site of areas of punctiform necrosis
slightly larger than those in Group II. Approximately 85% of the
tumor tissue persisted.
By contrast, 3 tumors showed areas of acidophilic necrosis.
There were no notable congestive phenomena, but the tumor
was surrounded by an inflammatory infiltrate consisting of histiocytes, plasmocytes, and a few rare polynuclear cells.
Group II-D (cf. Fig. 5). These 8 tumors received no HPD but
Fig. 1. Group l, acidophilic necrosis of tumor photocoagulated
with HPD. Hematoxylin, eosin, and safran; A, x 3.5; B, x 250.
JUNE
after sensitization
were photocoagulated. They never showed areas of total aci
dophilic necrosis. The tumor tissue was nevertheless destroyed,
95% necrotic, with isolated cellular elements with retracted nuclei
in the course of lysis. In one half of the cases, no neoplastic
glandular tubules remained in the periphery. In the other half,
there were a few glandular islets which persisted laterally.
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T. Patrice et al.
In 7 of 8 cases, the necrotic tumor was limited at its extremities
by a congested zone. The deep muscular layer was affected in
all cases.
DISCUSSION
Analysis of the results of this experimental study leads to the
following remarks.
(a) The comparison by the x2 test of Group I with Groups II-B,
II-C, and II-D combined shows a significant difference between
these 2 groups (x2 = 22.8; 1 d.f.).
The comparison of Group I with Groups II-B, II-C, and II-D
individually also has been done with the x2 test. After the
correcting Yates test, a significant difference between Group I
and each Group II is shown (x2 = 9.62; 1 d.f.).
(b) In the nontreated tumors, virtually no necrosis was seen
on the 12th day. In the mice receiving HPD only, very few
changes were seen within the tumors even when exposed to
light on the 10th day. By contrast, in the 2 series subjected to
either the action of the laser alone or the action of the laser after
injection of HPD, there was virtually total destruction of tumor
tissue.
The type of necrosis seen was not the same in Group I (laser
plus administration of HPD) and in Group II-D (laser only). When
the tumor had been previously sensitized by the administration
of HPD, there was total massive acidophilic necrosis with dis
appearance of all tissue or cell structures. This acidophilic necro
sis appeared to be different from the coagulation obtained by
the laser alone. Furthermore, the distribution of the few residual
glandular tubules is different in the 2 cases. After use of the laser
alone, the glandular tubules which were found only in one-half of
the cases studied were invariably distributed on either side of
the coagulation lesion and never at depth. The underlying mus
cular layer was affected. By contrast, when the tumor had been
previously sensitized with HPD (Group I), residual glands were
found in 3 of 4 cases and were distributed in a wreath-like
arrangement laterally as well as in depth. The underlying mus
cular layer was affected less frequently than that in Group II-D.
It appeared as if the destructive effect of the laser was potentialized by HPD with regard to the tumor itself but limited with
regard to the depth of the effects. In the absence of the admin
istration of HPD, congestive phenomena seen at the periphery
of the tumor tissue were more frequent and more marked.
It should be noted that HPD alone, in the absence of photocoagulation, had an effect on the tumor cells. Islets of punctiform
necrosis were seen in Group II-B. This was already reported in
vitro by Malik and Djaldetti (10) in 1980 when following the
administration of HPD scanning electron microscopy revealed
destruction of plasmic membranes and of the coat of Burkitttype tumor cells with inhibition of RNA and DNA synthesis.
This effect was increased with exposure to light (Group II-C),
and necrotic areas then were more extensive. Moan ef al. (11)
have emphasized the fact that the action of rays appears to be
more marked when HPD is intracellular.
Using tumor cell cultures, Kinsey ef al. (8) estimated that the
mass of cells destroyed was greater when the wavelength of the
light source was shorter. All of this did not seem to be corrobo
rated by our own study.
While there was no evidence to show that the laser lesions
were specific, there was nevertheless no similarity whatsoever
between the few areas of necrosis seen in the control series and
the total necrosis of tumors subject to photocoagulation.
2878
(c) Similar findings were reported by Dougherty ef al. (4) who
used a helium:neon or dye laser pumped by an argon laser to
destroy breast tumors. However, they did not make any study
of the histopathological lesions. Similarly, Thomson (14) obtained
tumor destruction (using an argon laser) of tumors sensitized
with acridine orange instead of HPD.
In summary, in answer to the question raised at the beginning
of this report, the following conclusions may be drawn: (a) The
Nd-YAG laser destroyed colonie adenocarcinoma-type tumors
grafted in the mouse when these tumors were previously sensi
tized with hematoporphyrin derivative; (b) The coagulation necro
sis observed after the use of the laser alone was replaced here
by acidophilic necrosis; (c) This lesion was in no way specific.
The same type of lesion was seen in nontreated lesions. Never
theless, the laser increased necrosis considerably, while this
increase did not occur when tumors sensitized by hematopor
phyrin were irradiated with ordinary light.
A doubt remains as far as the mechanism of action is con
cerned. From this point of view, this paper raises more questions
than it gives answers. Singlet oxygen production as mentioned
by Weishaupt ef al. (15) could be one of the mechanisms;
however, that would require an absorption of HPD at 1.06 ^m.
Although we believe it could exist, technical problems prevent
us from asserting it clearly and drawing a comparison with the
0.632-MiTi absorption. A direct alteration of HPD into a toxic
compound under the heating effect could be another mechanism.
Further studies are being conducted by our department to try to
provide an answer.
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den Stoffe. Muench. Med. Wochenschr., 7: 2042-2044,1903.
14. Thomson, S. H. Photodestruction of mouse epithelial tumors after oral acridine
orange and argon laser. Cancer Res., 34: 3124-3127,1974.
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VOL. 43
Laser Destruction of Non- and HPD-sensitized Tumors
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Fig. 3. Group II-B, rare areas of scanty punctiform necrosis after administration of HPD. Hematoxylin, eosin, and safran; A, x 2.2; B, x 250.
Fig. 4. Group II-C, areas of punctiform necrosis after administration of HPD followed by local exposures to light. Hematoxylin, eosin, and safran; A, x 2.7; B, x 250.
Fig. 5. Group II-D, necrosis of tumor photocoagulated by Nd-YAG laser. Hematoxylin, eosin, and safran; A, x 2.8; B, x 250.
2879
JUNE 1983
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Research.
Neodymium-Yttrium Aluminium Garnet Laser Destruction of
Nonsensitized and Hematoporphyrin Derivative-sensitized
Tumors
Thierry Patrice, Marie-Françoise Le Bodic, Louis Le Bodic, et al.
Cancer Res 1983;43:2876-2879.
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